1. Field of the Invention
The present invention relates to a process for the preparation of zero standard sera useful in immunoassay techniques.
2. Description of the Prior Art
Radioligand hormone assays have now become routine in hospital laboratories throughout the world. The key to the sensitivity of the test is the availability of hormone-free sera to be used as negative controls and also as diluents.
For example, standard controlled serum which contains no thyroxine is necessary in order to obtain the zero point for the standard curve used in radioimmunoassays or immunoradiometric assays or enzyme immunoassays. In addition, the use of zero standard control serum is also necessary to obtain accurate readings of hyperthyroid serum. Thus, when zero standard control serum is available, hyperthyroid serum can be diluted by a known factor with the zero standard control serum. T4 tests are then run by the above-identified procedures and compared with values obtained from standard curves. After the readings are obtained from these curves, dilution factors are applied to the readings. Accurate factors are only obtained when the zero standards have very low levels of hormones.
Eisentraut, U.S. Pat. No. 3,776,698, for example, prepares zero standard serum by contacting a quantity of acidified serum with a particulate inorganic crystalline sorbent material which is selected from the group consisting of phosphates, oxides, hydroxides, silicates, carbonates, aluminates, and sulfates, of the metallic elements in groups IA, IIA, IIIA, IIB and VIII of the periodic table.
In addition, two alternative sources of hormone-free serum are also presently used: (1) serum made by accumulation of a pool of serum from patients with hypopituitarism who are on replacement thyroxine therapy, or (2) serum made by extended high speed centrifugation of free carbon-adsorbed serum from a healthy population.
The use of free carbon, however, carries with it difficulties in time and equipment, when large volumes of serum are to be processed.
In order to be able to remove thyroid hormones (such as T3 and T4) from the serum, it is necessary to provide certain effective pore sizes in the treating carbon. Commercially available carbon is usually made by activating the same with steam at temperatures around 1000.degree. C. The activation probably removes tars and insertion materials trapped between the naturally layered structure of the carbon, thus allowing access to the interlayer regions. The gain in surface area which is obtained, however, results in sacrifice of the structural rigidity of the final product.
The mechanical forces imposed on mixing the carbon with serum at high speed centrifugation are sufficient to fragment the fragile activated carbon structure. The supernatant serum normally contains carbon fragments visible as the appearance of dark color. Removal of the tiny carbon fragments from the serum requires the addition of technical grade kaolin, which acts as a gathering agent, followed by further mixing and centrifugation. Sometimes, even colloidal flocculation is not satisfactory for complete removal, and filtration through a micron size membrane is required.
Large carbon granules (having a range of about 400 microns in diameter) show poor efficiency of thyroid hormone removal, whereas smaller powdered carbon granules, while more efficient, are far too slow in the absence of pressure.
An attempt to increase the rate of hormone removal, by artificially providing a pressure differential across a bed of carbon particles so as to increase the availability of the inside pores of small powdered carbon structure to the hormone molecules, runs into various problems. The structural rigidity of the carbon particles may be insufficient to withstand the applied pressure, and the pores may break with concommitant collapse of the particles. Also, channeling quickly occurs across the bed, followed by rapid loss in removal efficiency.
Carbon has been loaded on a sheet having particles homogeneously distributed and firmly retained therein, and having substantial freedom from ruboff, flaking and smudging, as described in Bodendorf et al, U.S. Pat. No. 3,149,023. The Bodendorf et al sheets, however, are used as cigarette filters, air filters, gas filters, wrappers for fruit and substances prone to discoloration or spoilage by gases in the atmosphere, deodorizer layers in laminated sheet products for sanitary napkins, and for surgical dressings for wounds, and the like. They are not used in the removal of thyroid hormones from serum.
The Bodendorf materials are not even described as being useful for high pressure applications and, most importantly, for high pressure applications in the treatment of a biological material such as serum, wherein the integrity and composition of the final product is critical.
In preparing zero standard serum, the final product should contain all the necessary natural serum components except the hormones. Inasmuch as possible, proteins, carbohydrates, lipids, vitamins, and salts should remain at substantially the same levels as prior to the treatment. There is no indication in Bodendorf et al that their carbon filled sheets can be used for the treatment of such delicate biological materials as human serum, that the treatments could be carried out at high pressures, and that they would result in selective removal of hormones.
In sum, a method for the removal of hormones from human serum to prepare zero standard serum which method is quick, inexpensive, and simple to operate, which uses materials which are simple to prepare, and which is nondestructive and non-modifying of the final standard product has not yet been described by the prior art.
A need therefore continues to exist for such a method, and the present invention fulfills that need.